Project Summary Inactivation of the p53 tumor suppression pathway is a pivotal event in the formation of most human cancers. Although p53-mediated cell-cycle arrest, senescence and apoptosis serve as critical barriers to cancer development, accumulating evidence suggests that loss of p53-dependent cell cycle arrest, apoptosis, and senescence is not sufficient to abrogate the tumor suppression activity of p53. Several mouse models suggest that tumor suppression by p53 can be achieved in the absence of those canonical functions. The p53 protein achieves diverse cellular outcomes by serving as a DNA-binding transcription factor that selectively modulates the expression of certain p53 transcriptional target genes. Numerous studies have established that acetylation of p53 acts as a key signal in promoter-specific activation of p53 target gene expression. Notably, our earlier studies demonstrate that the p533KR (3KR:K117R+K161R+K162R) acetylation-deficient mutant mouse model, which lacks the ability to undergo p53-mediated apoptosis, senescence and cell cycle arrest, are not significantly prone to developing tumors when compared to p53-null mice. Ferroptosis is a regulated form of non-apoptotic cell death driven by accumulation of lipid hydroperoxides. We found that p53 inhibits cystine uptake and sensitizes cells to ferroptosis by repressing expression of SLC7A11, a key component of the cystine/glutamate antiporter. Indeed, p533KR, an acetylation-defective mutant that fails to induce cell-cycle arrest, senescence and apoptosis, fully retains the ability to regulate SLC7A11 expression and promote ferroptosis. Nevertheless, it remains unclear how p53-mediated ferroptosis is regulated and whether p53- mediated ferroptosis is absolutely required for its remaining tumor suppression. In our preliminary studies, we have identified a novel p53 acetylation site at lysine K98 in mouse p53 (or K101 for human p53). While the loss of K98 acetylation (p53K98R) alone has very modest effects on p53-mediated transactivation, simultaneous mutations at all four acetylation sites (p534KR: K98R+K117R+K161R+K162R) completely abolish its ability to regulate metabolic targets such as TIGAR and SLC7A11. The central hypothesis to be tested here is whether p53 acetylation plays a major role in modulating p53 functions and whether p53-mediated ferroptosis is acts as a key mechanism in tumor suppression in the absence of cell-cycle arrest, apoptosis and senescence. In Aim 1, we will elucidate the molecular mechanisms of acetylation in modulating p53-mediated ferroptotic responses and tumor growth in human cancer cells. In Aim 2, we will examine the physiological significance of p53 acetylation as well as p53-mediated ferroptosis in vivo by establishing a new p53-mutant (p534KR/4KR) mouse model.